Field pea cultivar CDC 0001

ABSTRACT

A field pea cultivar designated CDC 0001 is disclosed. The invention relates to the seeds of field pea cultivar CDC 0001, to the plants of field pea cultivar CDC 0001, to plant parts of field pea cultivar CDC 0001 and to methods for producing a field pea plant produced by crossing field pea cultivar CDC 0001 with itself or with another field pea cultivar. The invention also relates to methods for producing a field pea plant containing in its genetic material one or more transgenes and to the transgenic field pea plants and plant parts produced by those methods. This invention also relates to field pea cultivars or breeding cultivars and plant parts derived from field pea cultivar CDC 0001, to methods for producing other field pea cultivars, lines or plant parts derived from field pea cultivar CDC 0001 and to the field pea plants, varieties, and their parts derived from use of those methods. The invention further relates to hybrid field pea seeds, plants and plant parts produced by crossing field pea cultivar CDC 0001 with another field pea cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive field pea, Pisumsativum (L) designated CDC 0001. All publications cited in thisapplication are herein incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single cultivar animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to drought andheat, and better agronomic quality.

Field pea (Pisum sativum L.), an annual legume, is also commonlyreferred to as pea and dry pea. Field pea most likely originated inSouthwest Asia and is widely grown in Russia, China, Canada, Europe,Australia and the United States. It is primarily used as a grain crop,for livestock feed or as a vegetable. Field pea is a grain legumecommonly used throughout the world in human cereal grain diets. Fieldpea is among the oldest crops in the world as it was cultivated as earlyas 9000 years ago.

The field pea is one of the world's important legume crops and is grownon over 25 million acres worldwide. Annual worldwide production isestimated at 2.5 million metric tons of dry beans harvested from 9million hectares. Plant breeders have played an important role in thedevelopment of the field pea industry as it exists in the United Statesand Canada today.

The protein in field pea seed is rich in the amino acids lysine andtryptophan as compared to cereal grains. Peas contain high levels ofcarbohydrates, are low in fiber and contain a large percentage of totaldigestable nutrients, which makes them excellent livestock feed. Also,field peas contain 5 to 20% less of the trypsin inhibitors than soybean.This allows it to be directly fed to livestock without having to gothrough the extrusion heating process.

Field pea is a cool-season annual herbaceous legume. There are two maintypes of field peas. One type has round seeds and is used primarily forfood and feed. The other type has wrinkled seeds and is usuallyharvested when immature and used for freezing and canning. Pea seeds mayhave either green or yellow cotyledons under a white or sometimes palegreen seed coat. Field pea seed weighs from 100 to 350 g/1000 seeds whendry and mature.

Most pea varieties produce white to reddish-purple flowers, which areself-pollinated. Each flower will produce a pod containing four to nineseeds. Pea varieties have either indeterminate or determinate floweringhabit. Determinate flowering varieties will flower for long periods andripening can be prolonged under cool, wet conditions. Indeterminatevarieties are later in maturity ranging from 90 to 100 days. Determinatevarieties will flower for a set period and ripen with earlier maturityof 80 to 90 days. Field peas are sensitive to heat stress at flowering,which can reduce pod and seed set.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of field pea plant breeding is to develop new, unique andsuperior field pea cultivars and hybrids. The breeder initially selectsand crosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selfing and mutations. The breeder has no direct control atthe cellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same field pea traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same cultivar twice by using theexact same original parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new field pea cultivars.

The development of new field pea cultivars requires the development andselection of field pea varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed is produced bymanual crosses between selected male-fertile parents or by using malesterility systems. These hybrids are selected for certain single genetraits such as pod color, flower color, pubescence color or herbicideresistance which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, field pea breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding. Enough seeds are harvested tomake up for those plants that did not germinate or produce seed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max L. Merr.) p 6.131-6.138 in S. J. O'Brien (ed)Genetic Maps: Locus Maps of Complex Genomes, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1993)) developed amolecular genetic linkage map that consisted of 25 linkage groups withabout 365 RFLP, 11 RAPD, three classical markers and four isozyme loci.See also, Shoemaker, R. C., RFLP Map of Soybean, p 299-309, in Phillips,R. L. and Vasil, I. K., eds. DNA-Based Markers in Plants, KluwerAcademic Press, Dordrecht, the Netherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet. 95:22-225, 1997.) SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. For example, molecularmarkers are used in soybean breeding for selection of the trait ofresistance to soybean cyst nematode, see U.S. Pat. No. 6,162,967. Themarkers can also be used to select toward the genome of the recurrentparent and against the markers of the donor parent. This procedureattempts to minimize the amount of genome from the donor parent thatremains in the selected plants. It can also be used to reduce the numberof crosses back to the recurrent parent needed in a backcrossingprogram. The use of molecular markers in the selection process is oftencalled Genetic Marker-Enhanced Selection or Marker-Assisted Selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Mutation breeding is another method of introducing new traits into fieldpea varieties. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation (such as X-rays, Gamma rays, neutrons,Beta radiation, or ultraviolet radiation), chemical mutagens (such asbase analogues like 5-bromo-uracil), antibiotics, alkylating agents(such as sulfur mustards, nitrogen mustards, epoxides, ethyleneamines,sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine,nitrous acid or acridines. Once a desired trait is observed throughmutagenesis the trait may then be incorporated into existing germplasmby traditional breeding techniques. Details of mutation breeding can befound in “Principles of Cultivar Development” by Fehr, MacmillanPublishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan et al., Theor. Appl. Genet., 77:889-892, 1989.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

SUMMARY OF THE INVENTION

According to the invention, there is provided a new field pea cultivardesignated CDC 0001. This invention thus relates to the seeds of fieldpea cultivar CDC 0001, to the plants of field pea cultivar CDC 0001 andto methods for producing a field pea plant produced by crossing thefield pea cultivar CDC 0001 with itself or another field pea cultivar,and the creation of variants by mutagenesis or transformation of fieldpea cultivar CDC 0001.

Thus, any such methods using the field pea cultivar CDC 0001 are part ofthis invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using field pea cultivarCDC 0001 as at least one parent are within the scope of this invention.Advantageously, this field pea cultivar could be used in crosses withother, different, field pea plants to produce first generation (F₁)field pea hybrid seeds and plants with superior characteristics.

In another aspect, the present invention provides for single or multiplegene converted plants of field pea cultivar CDC 0001. The transferredgene(s) may preferably be a dominant or recessive allele. Preferably,the transferred gene(s) will confer such traits as herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, male sterility, enhanced nutritional quality, andindustrial usage. The gene may be a naturally occurring field pea geneor a transgene introduced through genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of field pea plant CDC 0001. The tissue culturewill preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing fieldpea plant, and of regenerating plants having substantially the samegenotype as the foregoing field pea plant. Preferably, the regenerablecells in such tissue cultures will be embryos, protoplasts, meristematiccells, callus, pollen, leaves, anthers, roots, root tips, flowers,seeds, pods or stems. Still further, the present invention providesfield pea plants regenerated from the tissue cultures of the invention.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. Allele is any of one or more alternative forms of a gene, all ofwhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Cotyledon. A cotyledon is a type of seed leaf. The cotyledon containsthe food storage tissues of the seed.

Embryo. The embryo is the small plant contained within a mature seed.

Emergence. This score indicates the ability of the seed to emerge whenplanted 3″ deep in sand and with a controlled temperature of 25° C. Thenumber of plants that emerge each day are counted. Based on this data,each genotype is given a 1 to 9 score based on its rate of emergence andpercent of emergence. A score of 9 indicates an excellent rate andpercent of emergence, an intermediate score of 5 indicates averageratings and a 1 score indicates a very poor rate and percent ofemergence.

Hilum. This refers to the scar left on the seed which marks the placewhere the seed was attached to the pod prior to the seed beingharvested.

Hypocotyl. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

Lodging Resistance. Lodging resistance is measured on a subjective scaleof very good (VG), good (G), fair (F) and poor (P).

Phenotypic Score. The Phenotypic Score is a visual rating of generalappearance of the cultivar. All visual traits are considered in thescore including healthiness, standability, appearance and freedom fromdisease. Ratings are scored from 1 being poor to 9 being excellent.

Plant Height. Plant height is taken from the top of the soil to the topnode of the plant and is measured in centimeters.

Pod. This refers to the fruit of a field pea plant. It consists of thehull or shell (pericarp) and the field pea seeds.

Protein Percent. Field pea seeds contain a considerable amount ofprotein. Protein is generally measured by NIR spectrophotometry, and isreported on an as is percentage basis.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Relative maturity. “Early maturity” is defined as being around 80 days,“medium maturity” is defined as being 90-92 days and “late maturity” isdefined as 98 days and beyond.

Seed Protein Peroxidase Activity. Seed protein peroxidase activityrefers to a chemical taxonomic technique to separate cultivars based onthe presence or absence of the peroxidase enzyme in the seed coat. Thereare two types of field pea cultivars: those having high peroxidaseactivity (dark red color) and those having low peroxidase activity (nocolor).

Seeds per Pound. Field pea seeds vary in seed size, therefore, thenumber of seeds required to make up one pound also varies. This affectsthe pounds of seed required to plant a given area and can also impactend uses.

Single Gene Converted (Conversion). Single gene converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a cultivar are recovered inaddition to the single gene transferred into the cultivar via thebackcrossing technique or via genetic engineering.

Stem vine length. Stem vine length Is measured in centimeters from thestem of the plant to ground and is observed after flowering when podsare fully swollen.

Time of flowering. Time of flowering is observed when approximately 30%of plants have one flower open.

DETAILED DESCRIPTION OF THE INVENTION

Field pea cultivar CDC 0001 is a green field pea with medium-rangerelative maturity under Saskatoon, Saskatchewan, Canada locationconditions.

The cultivar has shown uniformity and stability, as described in thefollowing cultivar description information. It has been self-pollinateda sufficient number of generations with careful attention to uniformityof plant type. The cultivar has been increased with continuedobservation for uniformity.

Field pea cultivar CDC 0001 has the following morphologic and othercharacteristics (based primarily on data collected at Saskatoon,Saskatchewan, Canada).

TABLE 1 CULTIVAR DESCRIPTION INFORMATION Plant type: Green field peaPlant habit: Between determinate and interdeterminate (between tall andbushy) Plant height: Greater than 50 cm Stem vine length: 90 cm to 115cm Stem fasciation: Absent Presence of leaflets: Leafed Relativematurity: Medium (90–92 days) Flower color: White Flower color wing:White Flower shape of base: Level or straight Time of flowering: MediumProtein content: 25% Stipule: Development: Normal Marbling (beforeflowering): Present Maxium density of marbling: Sparse Pod: Length(observe at first flowering node): Medium Width (observe at firstflowering node): Medium Curvature (when fully swollen): Absent Color(immature): Light Green Color (when fully swollen): Green Parchment(when dry and papery): Entirely present Shape of distal part (when fullyswollen): Pointed Seed: Shape: Spherical Size: Medium Weight (g/1000seed): 230 Color of cotyledon: Green Black color of hilum: Absent Daysto maturity: 99 days Reaction to Diseases: Mycosphaerella blight(Mycosphaerella pinodes): Moderately susceptible Ascochyta root rot(Phoma medicaginis var. pinodella): Moderately susceptible Powderymildew (Erysiphe polygoni): Moderately susceptible

This invention is also directed to methods for producing a field peaplant by crossing a first parent field pea plant with a second parentfield pea plant, wherein the first or second field pea plant is thefield pea plant from the field pea cultivar CDC 0001. Further, bothfirst and second parent field pea plants may be from cultivar CDC 0001.Therefore, any methods using field pea cultivar CDC 0001 are part ofthis invention: selfing, backcrosses, hybrid breeding, and crosses topopulations. Any plants produced using field pea cultivar CDC 0001 as atleast one parent are within the scope of this invention.

Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues by using either microprojectile-mediated delivery with abiolistic device, or by using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

Field pea cultivar CDC 0001 is similar to field pea cultivar Nitouche.While similar to field pea cultivar Nitouche, there are significantdifferences including: field pea cultivar CDC 0001 has a sparse maximumdensity of marbling for its stipule while field pea cultivar Nitouchehas a very sparse maximum density of marbling for its stipule. Field peacultivar CDC 0001 has a pointed pod shape at the distal part while fieldpea cultivar Nitouche has a blunt pod shape at the distal part. Inaddition, field pea cultivar CDC 0001 is moderately susceptible topowdery mildew while field pea cultivar Nitouche is susceptible topowdery mildew.

Further Embodiments of the Invention

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed cultivar or line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid and can beused alone or in combination with other plasmids to provide transformedfield pea plants using transformation methods as described below toincorporate transgenes into the genetic material of the field peaplant(s).

Expression Vectors for Field Pea Transformation: Marker Genes

Expression vectors include at least one genetic marker operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase. II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, Imagene Green™, p. 1-4 (1993) and Naleway et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Field Pea Transformation: Promoters

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in a certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in field pea. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in field pea. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in field pea or the constitutive promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in field pea.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); PEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, Xba1/NcoI fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXba1/NcoI fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in field pea.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in field pea. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promotersuch as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine during protein synthesis and processing where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., PlantMol. Biol. 9:3-17 (1987); Lerneret al., Plant Physiol. 91:124-129(1989); Fontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal., Cell 39:499-509 (1984); Steifel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a field pea plant. Inanother preferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology, CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant cultivar can be transformed with one ormore cloned resistance genes to engineer plants that are resistant tospecific pathogen strains. See, for example Jones et al., Science266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Ptogene for resistance to Pseudomonas syringae pv. tomato encodes a proteinkinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2 genefor resistance to Pseudomonas syringae).

B. A gene conferring resistance to a pest, such as soybean cystnematode. See e.g., PCT Application WO96/30517; PCT ApplicationWO93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of aBt-δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genescan be purchased from American Type Culture Collection, Manassas, Va.,for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), of the nucleotide sequences of several Cliviaminiata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, variants thereof, mimetics based thereon, orantagonists or agonists thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., which discloses genes encoding insect-specific,paralytic neurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

T. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. Briggs, S. Current Biology, 5(2)(1995).

U. Antifungal genes. See Cornelissen and Melchers, Plant Physiol.,101:709-712 (1993); Parijs et al., Planta 183:258-264 (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998).

2. Genes That Confer Resistance to an Herbicide, For Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT, bar, genes), and pyridinoxy or phenoxy proprionicacids and cyclohexones (ACCase inhibitor-encoding genes). See, forexample, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses thenucleotide sequence of a form of EPSP which can confer glyphosateresistance. A DNA molecule encoding a mutant aroA gene can be obtainedunder ATCC accession number 39256, and the nucleotide sequence of themutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Europeanpatent application No. 0 333 033 to Kumada et al., and U.S. Pat. No.4,975,374 to Goodman et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean application No. 0 242 246 to Leemans et al. DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased phytate content—1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize for example, this could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene); Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene); Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase); Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes); Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Field Pea Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B. Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987);Sanford, J. C., Trends Biotech. 6:299 (1988); Klein et al.,Bio/Technology 6:559-563 (1988); Sanford, J. C., Physiol Plant 7:206(1990); Klein et al., Biotechnology 10:268 (1992). See also U.S. Pat.No. 5,015,580 (Christou, et al.), issued May 14, 1991; U.S. Pat. No.5,322,783 (Tomes, et al), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of field pea target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic cultivar. The transgenic cultivar could then becrossed with another (non-transformed or transformed) cultivar, in orderto produce a new transgenic cultivar. Alternatively, a genetic traitwhich has been engineered into a particular field pea line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite cultivar into anelite cultivar, or from a cultivar containing a foreign gene in itsgenome into a cultivar or cultivars which do not contain that gene. Asused herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Single Gene Conversions of Field Pea

When the term field pea plant is used in the context of the presentinvention, this also includes any single gene conversions of thatcultivar. The term single gene converted plant as used herein refers tothose field pea plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a cultivar are recovered inaddition to the single gene transferred into the cultivar via thebackcrossing technique. Backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into thecultivar. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, i.e.,crossing back 1, 2, 3, 4, 5, 6, 7, 8, 9 or more times to the recurrentparent. The parental field pea plant which contributes the gene for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental field pea plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman &Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second cultivar(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a fieldpea plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent, as determined at the 5%significance level when grown in the same environmental conditions.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalcultivar. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original cultivar. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new cultivar but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic; examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234 and 5,977,445.

Further reproduction of the cultivar can occur by tissue culture andregeneration. Tissue culture of various plant tissues and regenerationof plants therefrom is well known and widely published. For example,reference may be had to Komatsuda, T. et al., Crop Sci. 31:333-337(1991); Stephens, P. A., et al., Theor. Appl. Genet. (1991) 82:633-635;Komatsuda, T. et al., Plant Cell, Tissue and Organ Culture, 28:103-113(1992); Dhir, S. et al., Plant Cell Reports (1992) 11:285-289; Pandey,P. et al., Japan J. Breed. 42:1-5 (1992); and Shetty, K., et al., PlantScience 81:245-251 (1992); as well as U.S. Pat. No. 5,024,944 issuedJun. 18, 1991 to Collins et al., and U.S. Pat. No. 5,008,200 issued Apr.16, 1991 to Ranch et al. Thus, another aspect of this invention is toprovide cells which, upon growth and differentiation, produce field peaplants having the physiological and morphological characteristics offield pea cultivar CDC 0001.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. See also U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445; Brar, M. S., et al, In Vitro Cell. Deve. Biol.-Plant 35:8-12,January-February, 1999 and Brar, M. S., et al, In Vitro Cell. Deve.Biol.-Plant 35:222-225, May-June, 1999.

This invention also is directed to methods for producing a field peaplant by crossing a first parent field pea plant with a second parentfield pea plant wherein the first or second parent field pea plant is afield pea plant of field pea cultivar CDC 0001. Further, both first andsecond parent field pea plants can come from field pea cultivar CDC0001. Thus, any such methods using the field pea cultivar CDC 0001 arepart of this invention: selfing, backcrosses, hybrid production, crossesto populations, and the like. All plants produced using field peacultivar CDC 0001 as at least one parent are within the scope of thisinvention, including those developed from varieties derived from fieldpea cultivar CDC 0001. Advantageously, this field pea cultivar could beused in crosses with other, different, field pea plants to produce firstgeneration (F₁) field pea hybrid seeds and plants with superiorcharacteristics. The cultivar of the invention can also be used fortransformation where exogenous genes are introduced and expressed by thecultivar of the invention. Genetic variants created either throughtraditional breeding methods using field pea cultivar CDC 0001 orthrough transformation of field pea cultivar CDC 0001 by any of a numberof protocols known to those of skill in the art are intended to bewithin the scope of this invention.

The following describes breeding methods that may be used with field peacultivar CDC 0001 in the development of further field pea plants. Onesuch embodiment is a method for developing a field pea cultivar CDC 0001progeny field pea plant in a field pea plant breeding programcomprising: obtaining the field pea plant, or a part thereof, of fieldpea cultivar CDC 0001 utilizing said plant or plant part as a source ofbreeding material and selecting a field pea cultivar CDC 0001 progenyplant with molecular markers in common with field pea cultivar CDC 0001and/or with morphological and/or physiological characteristics selectedfrom the characteristics listed in Tables 1 or 2. Breeding steps thatmay be used in the field pea plant breeding program include pedigreebreeding, backcrossing, mutation breeding, and recurrent selection. Inconjunction with these steps, techniques such as RFLP-enhancedselection, genetic marker enhanced selection (for example SSR markers)and the making of double haploids may be utilized.

Another method involves producing a population of field pea cultivar CDC0001 progeny field pea plants, comprising crossing field pea cultivarCDC 0001 with another field pea plant, thereby producing a population offield pea plants, which, on average, derive 50% of their alleles fromfield pea cultivar CDC 0001. A plant of this population may be selectedand repeatedly selfed or sibbed with a field pea cultivar resulting fromthese successive filial generations. One embodiment of this invention isthe field pea cultivar produced by this method and that has obtained atleast 50% of its alleles from field pea cultivar CDC 0001.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the invention includes field peacultivar CDC 0001 progeny field pea plants comprising a combination ofat least two CDC 0001 traits selected from the group consisting of thoselisted in Tables 1 and 2 or the CDC 0001 combination of traits listed inthe Summary of the Invention, so that said progeny field pea plant isnot significantly different for said traits than field pea cultivar CDC0001 as determined at the 5% significance level when grown in the sameenvironmental conditions. Using techniques described herein, molecularmarkers may be used to identify said progeny plant as an CDC 0001progeny plant. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions. Once such acultivar is developed its value is substantial since it is important toadvance the germplasm base as a whole in order to maintain or improvetraits such as yield, disease resistance, pest resistance, and plantperformance in extreme environmental conditions.

Progeny of field pea cultivar CDC 0001 may also be characterized throughtheir filial relationship with field pea cultivar CDC 0001 , as forexample, being within a certain number of breeding crosses of field peacultivar CDC 0001. A breeding cross is a cross made to introduce newgenetics into the progeny, and is distinguished from a cross, such as aself or a sib cross, made to select among existing genetic alleles. Thelower the number of breeding crosses in the pedigree, the closer therelationship between field pea cultivar CDC 0001 and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4 or 5 breeding crosses of field pea cultivar CDC 0001.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which field pea plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,pods, leaves, roots, root tips, anthers, and the like.

The seed of field pea cultivar CDC 0001, the plant produced from theseed, the hybrid field pea plant produced from the crossing of thecultivar with any other field pea plant, hybrid seed, and various partsof the hybrid field pea plant can be utilized for human food, livestockfeed, and as a raw material in industry.

Tables

In Table 2, traits and characteristics of field pea cultivar CDC 0001are compared to several competing varieties of commercial field peas ofsimilar maturity found in the Canada Yield Trials in 2005. In the tablebelow, column 1 shows the variety, column 2 indicates the number ofyears in the test, column 3 indicates the average percent yield forthree southern locations as compared to Alfetta, column 4 indicates theaverage percent yield from two northern locations as compared toAlfetta, column 5 shows the vine length and column 6 shows the seedweight (g/1000 seeds). The Alfetta variety is the “control” or“standard” the other varieties are compared to. CDC 0007 is also knownas CDC Golden.

TABLE 2 PAIRED COMPARISONS Seed % yield for % yield for Vine WeightYears in South (3 North (2 length (g/1000 Variety Test locations)locations) (cm) seeds) CDC 0001 6 100 108 80 230 Alfetta 9 100 100 60290 CDC 0007 6 117 109 85 230 CDC Mozart 9 114 107 70 220 Nitouche 9 9497 75 250

Deposit Information

A deposit of the Alternative Seeds Strategies, Inc.. proprietary fieldpea cultivar designated CDC 0001 disclosed above and recited in theappended claims has been made with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Virginia 20110. The dateof deposit was Jan. 17, 2008. The deposit of 2500 seeds was taken fromthe same deposit maintained by Alternative Seeds Strategies, Inc., sinceprior to the filing date of this application. All restrictions upon thedeposit have been removed, and the deposit is intended to meet all ofthe requirements of 37 C.F.R. §1.801-1.809. The ATCC accession number isPTA-8890. The deposit will be maintained in the depository for a periodof 30 years, or 5 years after the last reguest, or for the effectivelife of the patent, whichever is longer, and will be replaced asnecessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationssuch as single gene modifications and mutations, somaclonal variants,variant individuals selected from large populations of the plants of theinstant cultivar and the like may be practiced within the scope of theinvention, as limited only by the scope of the appended claims.

1. A seed of field pea cultivar CDC 0001, wherein a representativesample of seed of said cultivar was deposited under ATCC Accession No.PTA-8890.
 2. A field pea plant, or a part thereof, produced by growingthe seed of claim
 1. 3. A tissue culture of regenerable cells producedfrom the plant of claim 2, wherein said cells of the tissue culture areproduced from a plant part selected from the group consisting of leaf,pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip,pistil, anther, flower, stem and pod.
 4. A protoplast produced from theplant of claim
 2. 5. A protoplast produced from the tissue culture ofclaim
 3. 6. A field pea plant regenerated from the tissue culture ofclaim 3, wherein the plant has all the morphological and physiologicalcharacteristics of cultivar CDC
 0001. 7. A method for producing an Flhybrid field pea seed, wherein the method comprises crossing the plantof claim 2 with a different field pea plant and harvesting the resultantF1 hybrid field pea seed.
 8. A hybrid field pea seed produced by themethod of claim
 7. 9. A hybrid field pea plant, or a part thereof,produced by growing said hybrid seed of claim
 8. 10. A method ofproducing an herbicide resistant field pea plant wherein the methodcomprises transforming the field pea plant of claim 2 with a transgenethat confers resistance to an herbicide selected from the groupconsisting of imidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 11. An herbicideresistant field pea plant produced by the method of claim
 10. 12. Amethod of producing an insect resistant field pea plant wherein themethod comprises transforming the field pea plant of claim 2 with atransgene that confers insect resistance.
 13. An insect resistant fieldpea plant produced by the method of claim
 12. 14. The field pea plant ofclaim 13, wherein the transgene encodes a Bacillus thuringiensisendotoxin.
 15. A method of producing a disease resistant field pea plantwherein the method comprises transforming the field pea plant of claim 2with a transgene that confers disease resistance.
 16. A diseaseresistant field pea plant produced by the method of claim
 15. 17. Amethod of producing a field pea plant with modified fatty acidmetabolism, modified carbohydrate metabolism, or decreased phytatecontent, wherein the method comprises transforming the field pea plantof claim 2 with a transgene encoding a protein selected from the groupconsisting of phytase, fructosyltransferase, levansucrase, α-amylase,invertase and starch branching enzyme or transforming a plant with anantisense gene of stearyl-ACP desaturase.
 18. A field pea plant havingmodified fatty acid metabolism, modified carbohydrate metabolism ormodified phytate content produced by the method of claim
 17. 19. Amethod of introducing a desired trait into field pea cultivar CDC 0001wherein the method comprises: (a) crossing a CDC 0001 plant,representative seed having been deposited under ATCC Accession No.PTA-8890, with a plant of another field pea cultivar that comprises adesired trait to produce progeny plants wherein the desired trait isselected from the group consisting of male sterility, herbicideresistance, insect resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, or decreased phytate content and resistance tobacterial disease, fungal disease or viral disease; (b) selecting one ormore progeny plants that have the desired trait to produce selectedprogeny plants; (c) crossing the selected progeny plants with the CDC0001 plants to produce backcross progeny plants; (d) selecting forbackcross progeny plants that have the desired trait and thephysiological and morphological characteristics of field pea cultivarCDC 0001 listed in Table 1 to produce selected backcross progeny plants;and (e) repeating steps (c) and (d) three or more times in succession toproduce selected fourth or higher backcross progeny plants that comprisethe desired trait and all of the physiological and morphologicalcharacteristics of field pea cultivar CDC 0001 listed in Table
 1. 20. Aplant produced by the method of claim 19 wherein the plant has thedesired trait and all of the physiological and morphologicalcharacteristics of field pea cultivar CDC 0001 listed in Table
 1. 21.The plant of claim 20 wherein the desired trait is herbicide resistanceand the resistance is conferred to an herbicide selected from the groupconsisting of imidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 22. The plant of claim 20wherein the desired trait is insect resistance and the insect resistanceis conferred by a transgene encoding a Bacillus thuringiensis endotoxin.23. The plant of claim 20 wherein the desired trait is modified fattyacid metabolism, modified carbohydrate metabolism, or decreased phytatecontent, and said desired trait is conferred by a nucleic acid encodinga protein selected from the group consisting of phytase,fructosyltransferase, levansucrase, α-amylase, invertase and starchbranching enzyme or transforming a plant with an antisense gene ofstearyl-ACP desaturase.